CN120905405A - Application of homozygous double V-type of SNP locus related to respiratory rate of hippocampus with abdomen expansion in breeding - Google Patents
Application of homozygous double V-type of SNP locus related to respiratory rate of hippocampus with abdomen expansion in breedingInfo
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Abstract
The invention discloses an application of homozygous double V-type of a respiratory rate-related SNP locus of a belly-expanding sea horse in breeding, and belongs to the technical field of aquatic breeding. Wherein, the double type is randomly formed by single type I, single type II and single type III, wherein, the bases at hspa gene 433 and 727 of the single type I are G, G respectively, the bases at hspa gene 433 and 727 of the single type II are G, A respectively, the bases at hspa gene 433 and 727 of the single type III are A, A respectively, the homozygous double V type formed by the single type III has obvious advantages in respiratory rate and hspa gene expression compared with other double types, and the homozygous double V type can be used for auxiliary breeding of high-temperature resistant new strain molecules of the hippocampus, and quickening the breeding process of high-temperature resistant fine varieties of the hippocampus.
Description
Technical Field
The invention relates to an application of homozygous double V-type of a respiratory rate-related SNP locus of a belly-expanding sea horse in breeding, belonging to the technical field of aquatic breeding.
Background
The optimal cultivation temperature of the ventral hippocampus is 18-20 ℃, and the survival rate of the ventral hippocampus is seriously affected by high-temperature weather in summer. At present, the artificially cultivated high-temperature-resistant strain of the bellied sea horse is lacking in China. Respiratory rate is an important index for evaluating the high temperature tolerance of the hippocampus. The research team has cultivated the high-temperature resistant strain F 2 generations of the hippocampus, and positioned respiratory rate-related SNP so as to accelerate the breeding process of high-temperature resistant fine varieties of the hippocampus.
At present, no report of application of the respiratory rate-related SNP locus of the ventral hippocampus to breeding exists, and similarly, the application of the respiratory rate-related SNP locus of the ventral hippocampus in breeding is similar to that of the respiratory rate-related SNP locus of the ventral hippocampus. However, the key genes of the body weight/body length/body thickness related SNP can only be related to the growth performance of the hippocampus, the key genes of the oscillometric width/eye diameter related SNP can only be related to the ingestion ability of the hippocampus, and the key genes cannot be related to the respiratory rate of the hippocampus, so that the gene can not be applied to screening of the high-temperature resistant strain of the hippocampus.
Disclosure of Invention
In order to solve the defects in the prior art, the invention provides the application of double-type SNP loci related to the respiratory rate of the ventral hippocampus in breeding so as to promote the auxiliary breeding of high-temperature-resistant new strain molecules of the ventral hippocampus and accelerate the breeding process of high-temperature-resistant fine varieties of the ventral hippocampus.
In order to achieve the above object, the present invention adopts the following technical scheme:
Use of homozygous double V at the respiratory rate-related SNP locus of the hippocampal, wherein the double forms are randomly formed of haplotype I, haplotype II, and haplotype III, wherein the bases at the hspa gene 433 and 727 of haplotype I are G, G, the bases at the hspa gene 433 and 727 of haplotype II are G, A, respectively, the bases at the hspa gene 433 and 727 of haplotype III are A, A, respectively, the nucleotide sequence of hspa gene is as shown in SEQ ID NO:1 or SEQ ID NO:2, 3 haplotypes randomly form homozygous double I type G 433G433G727G727, heterozygous double II type G 433G433G727A727, homozygous double III type G 433G433A727A727, heterozygous double IV type G 433A433A727A727, and homozygous double V type a 433A433A727A727 types of double types, respectively, the homozygous double V types having significant advantages in terms of respiratory rate and hspa gene relative expression compared to other 4 types of double V types, and the homozygous double V type can be used for the novel hippocampal sea horse-resistant molecular breeding.
The invention has the advantages that the correlation analysis is carried out on 2 SNP loci (respectively positioned at hspa gene 433 and 727) of the hippocampus hspa gene (encoding gene of heat shock protein 5) and the respiratory rate of the hippocampus, and the 2 SNP loci are found to be obviously related to the respiratory rate, wherein the respiratory rate of a single III type (A 433A727) individual is obviously reduced. By further carrying out respiratory rate and hspa gene expression comparison analysis on 5 types of homozygous double type I (DI), heterozygous double type II (DII), homozygous double type III (DIII), heterozygous double type IV (DIV) and homozygous double type V (DV), the respiratory rate of the ventral hippocampus and the expression of hspa genes are found to have obvious differences between the generation of high-resistance Wen Pinji F 2 and the common population, wherein the average respiratory rate of the ventral hippocampus of the homozygous double type V (DV) is obviously lower than that of the other 4 types of double types, the relative expression quantity of hspa genes is obviously higher than that of the other 4 types of double types, and the homozygous double type V (DV) can be used as a molecular marker related to the respiratory rate and applied to the molecular assisted breeding of the ventral hippocampus high-temperature resistant new variety of the ventral hippocampus so as to accelerate the breeding process of the ventral hippocampus high-temperature resistant fine variety.
Drawings
FIG. 1 is a graph showing the comparison of respiratory rate of the hippocampal normal population and refractory strain F 2 th generation 5 months of age;
FIG. 2 is a graph showing comparison of the relative expression amounts of hspa genes in 5-month-old hippocampus, 5 types of doubled types.
Detailed Description
The present invention will be described in detail with reference to the drawings and examples. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional Biochemical reagent companies.
1. Materials and methods
1. Experimental fish
The experimental fish is a general group of the belly-expanded sea horse (Wild Type group is hereinafter referred to as W group) and a high-temperature resistant strain F 2 generation (Mutant Type group is hereinafter referred to as M group), which are both 5 months old and have good growth and development. Stored in the eastern Shandong university hippocampal research center.
The average breath frequency of the 5 month old M population bred by the research team is significantly lower than that of the W population in the 12:00-18:00 high temperature period of each day (figure 1).
The W and M groups of the ventral hippocampus were each 200-tailed, cultivated in 2 40L glass jars, 200-tailed each, and connected to a central circulatory system with mechanical and biological filtration, uv sterilization and protein separators. The salinity is 31-32 per mill, the temperature is 18-19 ℃, the pH is 8.2-8.3, and the plastic plants are used as the attachment base. Three times daily feeding frozen furuncle were performed at 8:00, 12:00 and 16:00. After each feeding for 2 hours, the residual feed and the excrement in the glass jar are siphoned out.
2. Screening of SNP locus of heat shock protein 5 coding gene (hspa gene hereinafter)
By analyzing the sequencing data of the W-and M-populations of the hippocampus, two linkage disequilibrium SNP sites at hspa genes 443 and 727 with higher genetic diversity (PIC > 0.5) were identified (table 1).
TABLE 1 SNP site information
To further amplify SNP sites at hspa gene 433 and 727 by PCR reaction, primer sequences covering all coding regions of hspa gene were designed (Table 2).
TABLE 2 primer information
Cutting 1/3 dorsal fin of 400 pieces of the W-group and the M-group of the sea horse, extracting DNA by an alkaline lysis method, amplifying partial gene fragments of the W-group and the M-group by PCR, and sending qualified PCR products to Shanghai Biotechnology company (Shanghai China) for sequencing.
3. High temperature test
The 5 month old hippocampus, which is expanded in the abdomen, is put into the same glass jar for cultivation, the cultivation temperature is increased to 22 ℃, and other cultivation conditions are the same as before. The culture is carried out for 7 days, respiratory rate was counted 12:00 a day.
4. Real-time fluorescent quantitative PCR
After the high-temperature test, 5 ends of each SNP (single nucleotide polymorphism) genotype of the hippocampus, which is expanded, are randomly picked up, total RNA at the tail is extracted by RNAiso Plus, and genome DNA is removed from the RNA by using PRIME SCRIPT RT kit to synthesize cDNA (complementary deoxyribonucleic acid). Real-time quantitative PCR was performed in a CFX96 TouchTM real-time PCR detection system using SYBR Green Premix Ex Taq for qRT-PCR detection. Reference gene 18S was used as a control. The primers used are shown in Table 2. All experiments were performed in more than 3 replicates.
5. Statistical analysis
Data are expressed as mean ± sampling error, analyzed by SPSS STATISTICS 17.0.0 software using one-way variance method. Significance was at p <0.05 or p <0.01.
2. Results
1. SNP locus analysis of hippocampus japonicus hspa gene
Through detection, the nucleotide sequence of the W group hspa gene of the hippocampus, which is the subject of SEQ ID NO. 1, and the nucleotide sequence of the M group hspa5 gene, which is the subject of SEQ ID NO. 2. Correspondingly, the amino acid sequence of the W group hspa gene of the hippocampus, which is expanded, is shown as SEQ ID NO. 3, and the amino acid sequence of the M group hspa5 gene, which is expanded, is shown as SEQ ID NO. 4.
Comparing the hspa genes of the W group and the M group and the amino acid coded by the hspa genes, the coding region of the hspa genes of the M group shares 2 mutation sites, which are missense mutation, the codon change type of the G 433A433 site is GGG-AGG, the glycine (G) and the arginine (R) which are respectively coded at the 2 nd exon, the codon change type of the G 727A727 site is GAA-AAA, and the glutamic acid (E) and the lysine (K) which are respectively coded at the 3 rd exon.
A total of 3 haplotypes, haplotype I (HI, G 433G727), haplotype II (HII, G 433A727) and haplotype III (HIII, A 433A727), were detected in the W and M populations of the hippocampus, and the information about the 3 haplotypes is specifically shown in Table 3.
Table 33 haplotype information
As can be seen from Table 3, the W population is predominantly haplotype I, with a small number of haplotypes II, no haplotypes III, the M population is predominantly haplotypes III, with a small number of haplotypes II, no haplotypes I.
The W population randomly formed 2 haplotypes, homozygous double type I (DI, G 433G433G727G727) and heterozygous double type II (DII, G 433G433G727A727), with frequencies of 71% and 29% respectively, and the 2 types of double type information are shown in Table 4.
The two haplotypes (HII, HIII) of the M population randomly form 3 types of double types, namely homozygous double type III (DIII, G 433G433A727A727), heterozygous double type IV (DIV, G 433A433A727A727) and homozygous double type V (DV, A 433A433A727A727), and the occurrence frequencies are 22%, 43% and 35% respectively, and the information of the 3 types of double types is shown in the table 4.
Table 45 double information
2.5 Double respiratory rate analysis of the ventral hippocampus
The statistics of the average respiratory rate of 5 kinds of double 7 balances of the hippocampus, which is expanded, after the high-temperature test is finished are shown in Table 5.
TABLE 5 statistics of average respiratory rate of 5 kinds of double 7 balances of Hippocampus japonicus with abdomen expansion
Note that the number superscript lower case letter within the same column indicates that the difference is significant (p < 0.05).
As can be seen from Table 5, under high temperature conditions (22 ℃), the respiratory rate of 3 double forms (DIII, DIV, DV) of the M group was lower than that of 2 double forms (DI, DII) of the W group, wherein the respiratory rate of homozygous double form III (DIII) and heterozygous double form IV (DIV) of the M group was not significantly different from that of 2 double forms (DI, DII) of the W group (p > 0.05), and the respiratory rate of homozygous double form V (DV) of the M group was significantly different from that of 2 double forms (DI, DII) of the W group (p < 0.05).
3. Analysis of relative expression level of hspa Gene of 5 kinds of double types of Hippocampus japonicus
After the high-temperature test is finished, the relative expression amounts of hspa genes of 5 types of homozygous double type I (DI), heterozygous double type II (DII), homozygous double type III (DIII), heterozygous double type IV (DIV) and homozygous double type V (DV) of the hippocampus are 3.78+/-0.69, 3.80+/-0.61, 3.85+/-0.58, 4.12+/-0.73 and 5.82+/-0.96 respectively through detection. The relative expression amounts of hspa genes are compared with each other as shown in FIG. 2.
As can be seen from FIG. 2, the relative expression levels of DV type hspa genes in M populations were significantly higher than those of the other 4 double types (p < 0.05), and there was no significant difference in the relative expression levels of DI type, DII type, DIII type and DIV type hspa genes (p > 0.05).
3. Conclusion(s)
SNPs are DNA genetic polymorphisms caused by single nucleotide changes in the genome, are widely distributed in the genome, have stable inheritance, and are very important molecular markers. The invention identifies two linkage disequilibrium SNP loci at hspa gene 433 and 727 with higher genetic diversity (PIC > 0.5), which exist in 3 haplotypes (haplotype I, haplotype II and haplotype III). These 3 haplotypes randomly formed 5 haplotypes (homozygous double type I, heterozygous double type II, homozygous double type III, heterozygous double type IV and homozygous double type V). These two SNP sites of hspa gene lead to missense mutation of codons, and amino acid sequence change can lead to protein function change.
Respiratory rate is an important index for evaluating the growth of fishes at high temperature, and in order to verify whether mutation of two SNP loci of hspa genes is related to the respiratory rate of the abdominocentesis hippocampus, the invention identifies common population and high-temperature resistant strain F 2 generation, and the respiratory rate of different types of SNP is counted by 7-day high-temperature test. As a result, it was found that the respiratory rate of the randomly formed homozygous double V type (DV, A 433A433A727A727) of the haplotype III (HIII, A 433A727) of the refractory line F 2 generation was significantly advantageous compared to the normal population. It was thus concluded that simultaneous mutation at 433 and 727 (haplotype III SNP A 433A727) significantly reduced respiratory rate of the hippocampus at high temperature. Studies have shown that hspa gene is a member of heat shock protein 70 family and is closely related to the high temperature adaptation mechanism of fish. The glycine of the 2 nd exon of the single-fold type III SNP (A 433A727) of the high temperature resistant strain F 2 is changed into arginine, and the glutamic acid of the 3 rd exon is changed into lysine. The single SNP A 433A727 SNP mutation improves the expression of hspa gene, and hspa gene reduces oxidative damage by enhancing the activity of antioxidant enzyme, thereby maintaining the respiratory function of homozygous double V type (DV, A 433A433A727A727) of the high temperature resistant strain F 2 generation of the hippocampus. Therefore, the homozygote double V-type (DV, G 227G227A300A300 A433A433A727A727) of the ventral hippocampus shows stable respiratory rate at high temperature, and can be used for auxiliary breeding of high-temperature resistant novel variety molecules of the ventral hippocampus.
It should be noted that the above examples are only examples for clearly illustrating the present invention, and are not limiting to the embodiments of the present invention. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. Not all embodiments are exhaustive. Obvious changes and modifications which are extended by the technical proposal of the invention are still within the protection scope of the invention.
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Citations (5)
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Patent Citations (5)
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|---|---|---|---|---|
| US20020088017A1 (en) * | 1999-04-28 | 2002-07-04 | Board Of Regents, The University Of Texas System | Adenosine deaminase deficient transgenic mice and methods for the use thereof |
| CN101384621A (en) * | 2005-11-10 | 2009-03-11 | 受体生物公司 | Methods of generating receptor and ligand isoforms |
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